Abundant preclinical and epidemiologic data suggest that vitamin D possesses anti-neoplastic activity, and that individuals with higher plasma 25-hydroxyvitamin D [25(OH)D] levels have lower risk of colorectal cancer (CRC) and improved survival from CRC. Despite compelling evidence implicating vitamin D in CRC, critical questions remain about whether these findings reflect a true causal relationship, and what the biological mech- anisms of vitamin D activity are. Our central hypothesis is that vitamin D supplementation to achieve sufficient levels of 25(OH)D leads to improved survival in CRC patients and influences several neoplastic pathways that can be exploited as biomarkers of efficacy and targets for novel treatment strategies. We will test this hypothe- sis within two novel randomized clinical trials of vitamin D supplementation: a) a phase II double-blind random- ized trial of high- versus low-dose vitamin D3 in combination with chemotherapy in patients with metastatic CRC, and b) a randomized trial of preoperative high-dose vitamin D3 versus placebo in patients with resectable colon cancer for examination of vitamin D biology in fresh human tumor tissue. We will also lever- age a rich prospective cohort of metastatic CRC patients enrolled in a completed NCI-sponsored phase III trial of chemotherapy (CALGB/SWOG 80405) to further define and validate biomarkers of vitamin D action. In Aim 1, we will determine the impact of supplemental vitamin D on survival of CRC patients, test distinct hypotheses about the vitamin D dose-response relationship, and explore biomarkers of vitamin D status, response, and efficacy. We will investigate whether circulating levels of vitamin D binding protein and tumoral expression of vitamin D receptor, 1?-hydroxylase, and 24-hydroxylase are associated with improved survival within our ran- domized trials, and whether these markers modify the relationship between vitamin D and patient outcome. In Aim 2, we will conduct a precision medicine analysis of vitamin D to elucidate the mechanistic basis for its anti- tumor activity in CRC. We will explore the impact of vitamin D supplementation on tumoral markers of inflam- mation, lymphocytic infiltrates, and inflammatory gene signatures in surgical colectomy specimens from pa- tients treated with high-dose preoperative vitamin D3 versus placebo. We will also perform next generation se- quencing and Nanostring analysis of tumors from CRC patients with high- versus low vitamin D status to identi- fy unique genetic and transcriptional signatures associated with vitamin D-mediated carcinogenesis. In sum- mary, this translational proposal leverages innovative randomized trials of vitamin D to take the next critical steps in understanding vitamin D activity and biology in CRC. Our findings will confirm causality, provide new insights into biomarkers of vitamin D activity, and lead to potential inclusion of vitamin D into standard therapy ? including immunotherapy ? with the ultimate goal of improving the survival of patients with CRC.